STRUCTURAL DYNAMICS 2, 041709 (2015)
Possibilities for serial femtosecond crystallography sample delivery at future light sourcesa) L. M. G. Chavas,1,b) L. Gumprecht,1 and H. N. Chapman1,2,3,b) 1
Center for Free-Electron Laser Science, DESY, Notkestraße 85, 22607 Hamburg, Germany Department of Physics, University of Hamburg, Luruper Chaussee 149, 22607 Hamburg, Germany 3 Centre for Ultrafast Imaging, Luruper Chaussee 149, 22607 Hamburg, Germany 2
(Received 24 March 2015; accepted 6 May 2015; published online 14 May 2015)
Serial femtosecond crystallography (SFX) uses X-ray pulses from free-electron laser (FEL) sources that can outrun radiation damage and thereby overcome long-standing limits in the structure determination of macromolecular crystals. Intense X-ray FEL pulses of sufficiently short duration allow the collection of damage-free data at room temperature and give the opportunity to study irreversible time-resolved events. SFX may open the way to determine the structure of biological molecules that fail to crystallize readily into large well-diffracting crystals. Taking advantage of FELs with high pulse repetition rates could lead to short measurement times of just minutes. Automated delivery of sample suspensions for SFX experiments could potentially give rise to a much higher rate of obtaining complete measurements than at today’s third generation synchrotron radiation facilities, as no crystal alignment or complex robotic motions are required. This capability will also open up extensive time-resolved structural studies. New challenges arise from the resulting high rate of data collection, and in providing reliable sample delivery. Various developments for fully automated high-throughput SFX experiments are being considered for evaluation, including new implementations for a reliable yet flexible sample environment setup. Here, we review the different methods developed so far that best achieve sample delivery for X-ray FEL experiments and present some considerations towards the C 2015 goal of high-throughput structure determination with X-ray FELs. V Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License. [http://dx.doi.org/10.1063/1.4921220]
I. INTRODUCTION
The vast majority of high-resolution structures of macromolecules and macromolecular complexes have been determined by X-ray crystallography. All imaging methods that can attain molecular resolution use radiation that is energetic enough to ionize the sample, and with sufficient ionization events per given number of atoms (i.e., dose) the radiation can significantly degrade the structure under investigation. This problem of radiation damage can be overcome in X-ray crystallography by using well-ordered protein crystals with volumes greater than 1000 lm3, allowing the measurement of diffraction intensities within a tolerable dose of about 30 MGy when the sample is cooled to cryogenic temperatures (Henderson, 1990 and Owen et al., 2006). However, producing such large crystals is often not possible. Recently, a new approach to protein crystallography was demonstrated at X-ray free-electron laser (FEL) sources a)
Contributed paper, published as part of the 2nd International BioXFEL Conference, Ponce, Puerto Rico, January 2015. Authors to whom correspondence should be addressed. Electronic addresses: [email protected] and [email protected].
b)
2329-7778/2015/2(4)/041709/14
2, 041709-1
C Author(s) 2015 V
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Chavas, Gumprecht, and Chapman
Struct. Dyn. 2, 041709 (2015)
that vastly increases the tolerable dose. Sufficiently short femtosecond-duration pulses from an X-ray FEL can give rise to the formation of diffraction patterns before the onset of radiation damage (Neutze et al., 2000; Chapman et al., 2011; and Spence and Chapman, 2014). This technique, called serial femtosecond crystallography (SFX), consists of recording thousands to millions of single-crystal single-pulse diffraction patterns. Since the first SFX experiments (Chapman et al., 2011 and Boutet et al., 2012), the technique has been refined and successfully applied to various biological systems including in vivo grown crystals (Redecke et al., 2013), membrane proteins (Liu et al., 2013), and photo-induced samples (Tenboer et al., 2014). Phasing SFX data by anomalous diffraction have been demonstrated (Barends et al., 2014). The method of dispensing sample to the X-ray FEL beam depends on the size and quality of crystals available. For large crystals, a defocused pulse that imparts a dose of about 10 MGy creates a zone of destruction of approximately 25 lm diameter, and thus it is possible to collect short rotation series on a large frozen crystal by taking steps with larger spacing for each shot and rotation angle (Hirata et al., 2014). With large crystals, it is possible to obtain strong diffraction with a beam attenuated to levels where the crystal can survive several shots. Such an approach could be used for time-resolved structural studies if the dose is kept below conventional levels (radiochemical processes will develop during the dark time between shots, and cryogenic cooling will confer the same benefit as for measurements with synchrotron radiation). Although the relationship between beam size, dose, and the range of destruction has not been systematically studied, one can surmise that protein crystals smaller than about 50 lm diameter can only be measured with a single destructive pulse to outrun radiation damage. Various approaches have been developed and tried to deliver large numbers of small crystals of micrometer size or smaller to the beam, for the purpose of measuring a single diffraction pattern per shot. Under these conditions, there is no need to cryogenically cool the sample when using femtosecond X-ray pulses; samples can be measured at room temperature. The pulse vaporizes the exposed crystal, so data must be collected from a fresh crystal on each shot, and each exposure only gives a still diffraction pattern. A complete set of diffraction intensities can be acquired by collecting patterns in a serial fashion from a flowing sample, in which case each diffraction pattern is recorded at a random and unknown orientation of the crystal. This process is akin to powder diffraction. Indeed, if all patterns are summed together, a two-dimensional powder diffraction pattern is obtained. However, since the data are collected one grain at a time, it is possible to index each pattern and perform the summation on reflections of like Miller indices. In this way, a set of three-dimensional structure factors are obtained, where each is averaged over the same set of crystal shapes and sizes and beam fluctuations that would have produced a (nonoverlapping) Debye-Scherrer ring. This analysis method is referred to as Monte Carlo integration (Kirian et al., 2011; White et al., 2012; and White et al., 2013). Various sample delivery systems have been implemented for experiments at the Linac Coherent Light Source (LCLS) in California and the SPring-8 Angstrom Compact free-electron LAser (SACLA) in Japan. The two main classes of methods that have been demonstrated so far are flowing suspensions of crystals in a liquid or viscous medium across the beam and substrates or membranes supporting crystals that are rastered across the beam. With small or weakly diffracting crystals, it is crucial to keep the thickness of the supporting substrate or carrying medium to values that are not considerably larger than the crystals themselves, so that high-resolution reflections may be measured above the background generated by the medium. The first experiments at LCLS utilized gas-focused liquid jets (DePonte et al., 2008) of submicron crystals suspended in their buffer solution. These jets are typically 1 to 5 lm in diameter. A nozzle that extrudes a viscous medium such as lipidic cubic phase (LCP) has also been successfully used to deliver to the beam membrane proteins that crystallize in that medium (Weierstall et al., 2014). While producing higher background than liquid jets, the membrane crystals studied so far in LCP tended to be larger than 10 lm in diameter. The low flow rate of the LCP jet gives rise to about a 20-fold reduction in sample consumption for the same number of patterns collected with liquid jets. A low-flow extrusion of a grease matrix has also been tried (Sugahara et al., 2015). Electrospinning jets have lower flow rates than liquid jets and also consume less material compared with gas-focused liquid jets (Sierra et al., 2012). While
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Struct. Dyn. 2, 041709 (2015)
not yet tested extensively with crystals, aerosol beams created using aerodynamic lenses (Liu et al., 1995) have been used to collect coherent diffraction patterns from non-crystalline samples such as nanoparticles (Bogan et al., 2008), soot (Loh et al., 2012), cell organelles (van der Schot et al., 2015), and viruses (Seibert et al., 2011). A new innovation of a compact aerosol injector with improved particle focusing is described by Kirian et al. (2015). There are many ways to design experiments, and the field is still young and evolving. It is helpful to consider the priorities of an experimental design for serial crystallography, which are to (1) maximize diffraction data quality, especially on small samples; (2) maximize diffraction data collected per quantity of sample; and (3) maximize the experimental capacity by minimizing time for data collection and any down time. Since these guiding principles can in some situations be contradictory, or be quite dependent on the sample properties, a prudent design would maximize the diversity of data collection techniques and ideally be flexible enough to accommodate new improvements. While many groups are steadily improving the reliability of various delivery systems, and introducing new innovations, experiments often have to contend with jets that vary in direction or other jetting properties with time, and with wide ranges of the composition and viscosity of the jet medium. These problems often lead to loss of measurement time, as the injection system needs to be replaced and re-aligned. We review here different sample delivery methods developed for experimenting at X-ray FELs and describe concepts for a sample environment that could facilitate automation in data collection, sample screening, and nozzle exchange, which may allow SFX experiments to be carried out at high efficiency. II. SERIAL CRYSTALLOGRAPHY WITH PULSED SOURCES
The three guiding principles for SFX experiments listed above must necessarily be followed within the boundaries imposed by the characteristics of X-ray FEL sources and the limitations of instrumentation. Some relevant properties of current and pending X-ray FEL (XFEL) facilitates are summarized in Table I. As is apparent from this table, the experimental conditions at sources with repetition rates of 120 Hz and lower are considerably different to the European XFEL, which delivers bursts of pulses at up to 4.5 MHz, and LCLS II, which is proposed to operate at 1 MHz for photon energies up to 5 keV. The pulse rate dictates the frame rate of detectors required for SFX as well as the required refresh speed of sample delivery. At LCLS and SACLA, the camera readouts match the pulse deliveries, with the Cornell-SLAC Pixel Array Detector (CSPAD; Hart et al., 2012) and the Multi-Port Charge-Coupled Device (MPCCD; Kameshima et al., 2014) optimized to record images at 120 Hz and 60 Hz, respectively. With the Adaptive Gain Integrating Pixel Detector (AGIPD; Becker and Graafsma, 2012), it will be possible to store 352 frames per TABLE I. Principal properties to be considered for data collection, sample delivery, and characterization during SFX experiments at various hard X-ray FEL sources. The minimum sample speed is calculated assuming a required gap of 100 lm between pulses and assuming the source running at its highest-possible rate.
LCLS
LCLS II (1 000 1
10 000 10
2700 6
Yes …
No Yes (limited area)
Tape
Solid or liquid
10
100
60
…
No
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200-nm thick silicon nitride membrane. The wafer membrane was coated with crystals and placed in the vacuum environment for collection of diffraction patterns using the focused LCLS unmonochromatized pulses. To prevent desiccation of the sample in the vacuum chamber, the crystals were removed from their buffer and resuspended in oil before this solution was painted onto the membranes. By continuously raster-scanning the wafer, SFX data could be recorded at 10 Hz and hit fractions above 38% could be achieved, which should allow a full dataset to be acquired with less than 1 mg of protein (Hunter et al., 2014). The method is well suited for measuring diffraction from two-dimensional membrane crystals (Pedrini et al., 2014) and is suitable for time-resolved measurements. Sample consumption could possibly be reduced further by using structured wafers where crystals become trapped in regularly spaced wells (Zarrine-Afsar et al., 2012) or microfluidic chips (Heymann et al., 2014). It should be possible to acquire data at 120 Hz if the array was moved at a speed greater than about 12 mm/s (see Table I) which perhaps could be performed without interruptions if the sample was only scanned in one direction, for example, as a thin tape (Roessler et al., 2013). C. Aerosol particle injectors
Single particle diffraction experiments with X-ray FEL pulses were envisioned to be carried out on particles in the gas phase, without any substrate or carrying medium that would create a background that may overwhelm the desired signal (Neutze et al., 2000). Sample delivery technologies from mass spectroscopy were considered, and the first single-particle measurements at the Free-electron LASer in Hamburg (FLASH) were carried out using an aerodynamic lens (Bogan et al., 2008). The aerosol is first created from the sample suspension at atmospheric pressure using a nebulizer such as an electrospray aerosol generator or a gas-focused liquid jet and introduced into the vacuum environment of the experiment through a series of skimmers, apertures, and pumping stages. The coaxial lens apertures tend to drive particles toward the central streamline by balancing particle inertial forces and drag forces. Lens stacks are thus optimized for a particular range of particle sizes, and for particles with diameters of about 100 nm, collimated beams of tens of microns can be achieved. The speed of the particles ranges from about 5 to 20 m/s. An aerodynamic lens was used for single-particle diffraction of virus particles (Seibert et al., 2011), soot (Loh et al., 2012), particle dimers (Starodub et al., 2012), and cell organelles (Hantke et al., 2014). High particle hit rates have been achieved, although the fraction of those hits that occur in the most intense part of the focused beam is lower. Thus, the hit fraction depends on the X-ray beam profile in the focus, which tends to have wide low-intensity wings, and the threshold at which a pattern is counted as a hit. It might be feasible to inject sub-micron sized crystals by this approach, which could benefit in collecting data with less background at lower energies. Very recently, a compact aerosol injector was demonstrated that drives particles towards a focus of dimensions less than 10 lm diameter using a converging conical nozzle (Kirian et al., 2015). The injector is comparable in size to the gas-focused liquid jets described below, and hence could be interchangeably mounted with those. This method utilizes only the one orifice and thus gas is emitted at high speed, imparting velocities to the particles above 250 m/s. The higher the particle speed, the lower the particle hit rate for a given X-ray pulse rate (since the particle density is reduced), but this injector is currently the only one with high enough speeds for the 4.5 MHz burst rate of the European XFEL. D. Gas-focused liquid jets
The gas-focused dynamic virtual nozzle (GDVN) creates a jet of liquid that is typically a few microns in diameter moving at a velocity of several meters per second, injected into vacuum. Most SFX experiments to date have utilized GDVNs to deliver liquid suspensions of crystals to the beam. The nozzle consists of a hollow capillary usually made of glass and centered within a larger capillary tube. High-pressure gas flows through the interstitial space between the capillaries and focuses the liquid jet coming out of the inner capillary to a diameter that is much smaller than the orifice of that capillary (DePonte et al., 2008). This avoids clogging of
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the delivered particles that would occur by simply using a small orifice without gas focusing. Jet diameters of 1–5 lm are typically achieved, depending on the capillary diameters, the nozzle design, and the liquid and gas flows. Jet diameters as small as 300 nm have been demonstrated with a modified design where the orifice of the liquid line occurs at the point of the gas expansion into vacuum (DePonte et al., 2009). The emitted liquid forms a cylindrical column that eventually breaks up into drops. The breakup can be driven at a fixed frequency so that droplets are timed to the X-ray pulse arrival (Shapiro et al., 2008). However, since the drop diameter is about twice the column diameter, a lower background is achieved by placing the X-ray focus in the column. Particles and crystals that are elongated tend to align their long axis in the direction of the flow in the column and lose that alignment in the breakup region. It has been the practice so far to run the jet continuously rather than pulsing it to match the X-ray repetition rate. As such, the liquid flow rate is typically greater than 10 ll min 1 and tens of milligrams of microcrystals are often required for a full data set. Photoactivated samples can be studied by laser-pumping the sample at the required time before the X-ray pulse arrival, which is usually done by illuminating the entire jet with the pump beam. However, for delays longer than about 100 ls, it may be necessary to illuminate the sample inside the nozzle (Kupitz et al., 2014). By introducing another liquid capillary inserted in the first liquid line, it is possible to make a mixing jet (Wang et al., 2014). Here, the sample can mix diffusively with a substrate in the laminar flow of the jet at a well-defined time before X-ray exposure, which can be varied by a clever telescopic design. GDVNs have also been made by applying the soft lithography techniques of microfluidics (Trebbin et al., 2014). This has the advantage of easier mass production and in employing many designs for mixing and pulsing of jets. E. Electrospun jets
Instead of gas focusing, electrospinning forms a small jet in vacuum or atmosphere by application of high voltage (between the liquid and an external electrode) that creates a Taylor cone. Samples can consist of particles embedded in a viscoelastic polymer or in a conductive liquid of the right viscosity (Sierra et al., 2012 and Bogan et al., 2008). Slower velocities than liquid jets can be attained, and voltage gives an extra degree of freedom that allows higher velocity for a given sample flow rate (producing sub-micron jets). Typical flow rates are 0.14 to 3.1 ll min 1. No effect of the high electric field on the sample has been observed in protein structures obtained using this delivery mechanism (Kern et al., 2013). It is worth to note that this method is not suitable for measurements with synchrotron radiation since it was observed (in unpublished experiments at the Swiss Light Source by Bogan and Chapman) that the X-ray beam disrupts the formation of the Taylor cone, possibly due to ionization of the rest gas. F. Extruded viscous flows
The LCP extrusion nozzle is similar to the GDVN except that the sample is embedded in a high-viscosity medium that is pushed out of a nozzle at high pressure (Weierstall et al., 2014). This has primarily been used to dispense membrane proteins, such as G-protein coupled receptors, that crystallize in LCP (Liu et al., 2013 and Weierstall, 2014). The diameter of the extruded material matches the nozzle diameter (about 20 to 50 lm). Coaxial gas is used to stabilize the flow direction, and the method works in vacuum or air (Botha et al., 2015 and Nogly et al., 2015). The velocity of the medium is much slower than that of liquid jets and can be varied between about 25 to 100 mm s 1. This conserves sample and with the 120 Hz repetition rate of LCLS it has been possible to run the jet at less than 100 lm between shots, essentially hitting every crystal that flows by with an X-ray FEL repetition rate of 60 Hz. At the European XFEL, it would only be possible to utilize such jets with a single X-ray pulse per pulse train, that is, at 10 Hz. Due to the required high pressure, the reservoir must be located close to the nozzle, but the reservoir volume only needs be
Possibilities for serial femtosecond crystallography sample delivery at future light sourcesa) L. M. G. Chavas,1,b) L. Gumprecht,1 and H. N. Chapman1,2,3,b) 1
Center for Free-Electron Laser Science, DESY, Notkestraße 85, 22607 Hamburg, Germany Department of Physics, University of Hamburg, Luruper Chaussee 149, 22607 Hamburg, Germany 3 Centre for Ultrafast Imaging, Luruper Chaussee 149, 22607 Hamburg, Germany 2
(Received 24 March 2015; accepted 6 May 2015; published online 14 May 2015)
Serial femtosecond crystallography (SFX) uses X-ray pulses from free-electron laser (FEL) sources that can outrun radiation damage and thereby overcome long-standing limits in the structure determination of macromolecular crystals. Intense X-ray FEL pulses of sufficiently short duration allow the collection of damage-free data at room temperature and give the opportunity to study irreversible time-resolved events. SFX may open the way to determine the structure of biological molecules that fail to crystallize readily into large well-diffracting crystals. Taking advantage of FELs with high pulse repetition rates could lead to short measurement times of just minutes. Automated delivery of sample suspensions for SFX experiments could potentially give rise to a much higher rate of obtaining complete measurements than at today’s third generation synchrotron radiation facilities, as no crystal alignment or complex robotic motions are required. This capability will also open up extensive time-resolved structural studies. New challenges arise from the resulting high rate of data collection, and in providing reliable sample delivery. Various developments for fully automated high-throughput SFX experiments are being considered for evaluation, including new implementations for a reliable yet flexible sample environment setup. Here, we review the different methods developed so far that best achieve sample delivery for X-ray FEL experiments and present some considerations towards the C 2015 goal of high-throughput structure determination with X-ray FELs. V Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License. [http://dx.doi.org/10.1063/1.4921220]
I. INTRODUCTION
The vast majority of high-resolution structures of macromolecules and macromolecular complexes have been determined by X-ray crystallography. All imaging methods that can attain molecular resolution use radiation that is energetic enough to ionize the sample, and with sufficient ionization events per given number of atoms (i.e., dose) the radiation can significantly degrade the structure under investigation. This problem of radiation damage can be overcome in X-ray crystallography by using well-ordered protein crystals with volumes greater than 1000 lm3, allowing the measurement of diffraction intensities within a tolerable dose of about 30 MGy when the sample is cooled to cryogenic temperatures (Henderson, 1990 and Owen et al., 2006). However, producing such large crystals is often not possible. Recently, a new approach to protein crystallography was demonstrated at X-ray free-electron laser (FEL) sources a)
Contributed paper, published as part of the 2nd International BioXFEL Conference, Ponce, Puerto Rico, January 2015. Authors to whom correspondence should be addressed. Electronic addresses: [email protected] and [email protected].
b)
2329-7778/2015/2(4)/041709/14
2, 041709-1
C Author(s) 2015 V
041709-2
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Struct. Dyn. 2, 041709 (2015)
that vastly increases the tolerable dose. Sufficiently short femtosecond-duration pulses from an X-ray FEL can give rise to the formation of diffraction patterns before the onset of radiation damage (Neutze et al., 2000; Chapman et al., 2011; and Spence and Chapman, 2014). This technique, called serial femtosecond crystallography (SFX), consists of recording thousands to millions of single-crystal single-pulse diffraction patterns. Since the first SFX experiments (Chapman et al., 2011 and Boutet et al., 2012), the technique has been refined and successfully applied to various biological systems including in vivo grown crystals (Redecke et al., 2013), membrane proteins (Liu et al., 2013), and photo-induced samples (Tenboer et al., 2014). Phasing SFX data by anomalous diffraction have been demonstrated (Barends et al., 2014). The method of dispensing sample to the X-ray FEL beam depends on the size and quality of crystals available. For large crystals, a defocused pulse that imparts a dose of about 10 MGy creates a zone of destruction of approximately 25 lm diameter, and thus it is possible to collect short rotation series on a large frozen crystal by taking steps with larger spacing for each shot and rotation angle (Hirata et al., 2014). With large crystals, it is possible to obtain strong diffraction with a beam attenuated to levels where the crystal can survive several shots. Such an approach could be used for time-resolved structural studies if the dose is kept below conventional levels (radiochemical processes will develop during the dark time between shots, and cryogenic cooling will confer the same benefit as for measurements with synchrotron radiation). Although the relationship between beam size, dose, and the range of destruction has not been systematically studied, one can surmise that protein crystals smaller than about 50 lm diameter can only be measured with a single destructive pulse to outrun radiation damage. Various approaches have been developed and tried to deliver large numbers of small crystals of micrometer size or smaller to the beam, for the purpose of measuring a single diffraction pattern per shot. Under these conditions, there is no need to cryogenically cool the sample when using femtosecond X-ray pulses; samples can be measured at room temperature. The pulse vaporizes the exposed crystal, so data must be collected from a fresh crystal on each shot, and each exposure only gives a still diffraction pattern. A complete set of diffraction intensities can be acquired by collecting patterns in a serial fashion from a flowing sample, in which case each diffraction pattern is recorded at a random and unknown orientation of the crystal. This process is akin to powder diffraction. Indeed, if all patterns are summed together, a two-dimensional powder diffraction pattern is obtained. However, since the data are collected one grain at a time, it is possible to index each pattern and perform the summation on reflections of like Miller indices. In this way, a set of three-dimensional structure factors are obtained, where each is averaged over the same set of crystal shapes and sizes and beam fluctuations that would have produced a (nonoverlapping) Debye-Scherrer ring. This analysis method is referred to as Monte Carlo integration (Kirian et al., 2011; White et al., 2012; and White et al., 2013). Various sample delivery systems have been implemented for experiments at the Linac Coherent Light Source (LCLS) in California and the SPring-8 Angstrom Compact free-electron LAser (SACLA) in Japan. The two main classes of methods that have been demonstrated so far are flowing suspensions of crystals in a liquid or viscous medium across the beam and substrates or membranes supporting crystals that are rastered across the beam. With small or weakly diffracting crystals, it is crucial to keep the thickness of the supporting substrate or carrying medium to values that are not considerably larger than the crystals themselves, so that high-resolution reflections may be measured above the background generated by the medium. The first experiments at LCLS utilized gas-focused liquid jets (DePonte et al., 2008) of submicron crystals suspended in their buffer solution. These jets are typically 1 to 5 lm in diameter. A nozzle that extrudes a viscous medium such as lipidic cubic phase (LCP) has also been successfully used to deliver to the beam membrane proteins that crystallize in that medium (Weierstall et al., 2014). While producing higher background than liquid jets, the membrane crystals studied so far in LCP tended to be larger than 10 lm in diameter. The low flow rate of the LCP jet gives rise to about a 20-fold reduction in sample consumption for the same number of patterns collected with liquid jets. A low-flow extrusion of a grease matrix has also been tried (Sugahara et al., 2015). Electrospinning jets have lower flow rates than liquid jets and also consume less material compared with gas-focused liquid jets (Sierra et al., 2012). While
041709-3
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Struct. Dyn. 2, 041709 (2015)
not yet tested extensively with crystals, aerosol beams created using aerodynamic lenses (Liu et al., 1995) have been used to collect coherent diffraction patterns from non-crystalline samples such as nanoparticles (Bogan et al., 2008), soot (Loh et al., 2012), cell organelles (van der Schot et al., 2015), and viruses (Seibert et al., 2011). A new innovation of a compact aerosol injector with improved particle focusing is described by Kirian et al. (2015). There are many ways to design experiments, and the field is still young and evolving. It is helpful to consider the priorities of an experimental design for serial crystallography, which are to (1) maximize diffraction data quality, especially on small samples; (2) maximize diffraction data collected per quantity of sample; and (3) maximize the experimental capacity by minimizing time for data collection and any down time. Since these guiding principles can in some situations be contradictory, or be quite dependent on the sample properties, a prudent design would maximize the diversity of data collection techniques and ideally be flexible enough to accommodate new improvements. While many groups are steadily improving the reliability of various delivery systems, and introducing new innovations, experiments often have to contend with jets that vary in direction or other jetting properties with time, and with wide ranges of the composition and viscosity of the jet medium. These problems often lead to loss of measurement time, as the injection system needs to be replaced and re-aligned. We review here different sample delivery methods developed for experimenting at X-ray FELs and describe concepts for a sample environment that could facilitate automation in data collection, sample screening, and nozzle exchange, which may allow SFX experiments to be carried out at high efficiency. II. SERIAL CRYSTALLOGRAPHY WITH PULSED SOURCES
The three guiding principles for SFX experiments listed above must necessarily be followed within the boundaries imposed by the characteristics of X-ray FEL sources and the limitations of instrumentation. Some relevant properties of current and pending X-ray FEL (XFEL) facilitates are summarized in Table I. As is apparent from this table, the experimental conditions at sources with repetition rates of 120 Hz and lower are considerably different to the European XFEL, which delivers bursts of pulses at up to 4.5 MHz, and LCLS II, which is proposed to operate at 1 MHz for photon energies up to 5 keV. The pulse rate dictates the frame rate of detectors required for SFX as well as the required refresh speed of sample delivery. At LCLS and SACLA, the camera readouts match the pulse deliveries, with the Cornell-SLAC Pixel Array Detector (CSPAD; Hart et al., 2012) and the Multi-Port Charge-Coupled Device (MPCCD; Kameshima et al., 2014) optimized to record images at 120 Hz and 60 Hz, respectively. With the Adaptive Gain Integrating Pixel Detector (AGIPD; Becker and Graafsma, 2012), it will be possible to store 352 frames per TABLE I. Principal properties to be considered for data collection, sample delivery, and characterization during SFX experiments at various hard X-ray FEL sources. The minimum sample speed is calculated assuming a required gap of 100 lm between pulses and assuming the source running at its highest-possible rate.
LCLS
LCLS II (1 000 1
10 000 10
2700 6
Yes …
No Yes (limited area)
Tape
Solid or liquid
10
100
60
…
No
041709-7
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Struct. Dyn. 2, 041709 (2015)
200-nm thick silicon nitride membrane. The wafer membrane was coated with crystals and placed in the vacuum environment for collection of diffraction patterns using the focused LCLS unmonochromatized pulses. To prevent desiccation of the sample in the vacuum chamber, the crystals were removed from their buffer and resuspended in oil before this solution was painted onto the membranes. By continuously raster-scanning the wafer, SFX data could be recorded at 10 Hz and hit fractions above 38% could be achieved, which should allow a full dataset to be acquired with less than 1 mg of protein (Hunter et al., 2014). The method is well suited for measuring diffraction from two-dimensional membrane crystals (Pedrini et al., 2014) and is suitable for time-resolved measurements. Sample consumption could possibly be reduced further by using structured wafers where crystals become trapped in regularly spaced wells (Zarrine-Afsar et al., 2012) or microfluidic chips (Heymann et al., 2014). It should be possible to acquire data at 120 Hz if the array was moved at a speed greater than about 12 mm/s (see Table I) which perhaps could be performed without interruptions if the sample was only scanned in one direction, for example, as a thin tape (Roessler et al., 2013). C. Aerosol particle injectors
Single particle diffraction experiments with X-ray FEL pulses were envisioned to be carried out on particles in the gas phase, without any substrate or carrying medium that would create a background that may overwhelm the desired signal (Neutze et al., 2000). Sample delivery technologies from mass spectroscopy were considered, and the first single-particle measurements at the Free-electron LASer in Hamburg (FLASH) were carried out using an aerodynamic lens (Bogan et al., 2008). The aerosol is first created from the sample suspension at atmospheric pressure using a nebulizer such as an electrospray aerosol generator or a gas-focused liquid jet and introduced into the vacuum environment of the experiment through a series of skimmers, apertures, and pumping stages. The coaxial lens apertures tend to drive particles toward the central streamline by balancing particle inertial forces and drag forces. Lens stacks are thus optimized for a particular range of particle sizes, and for particles with diameters of about 100 nm, collimated beams of tens of microns can be achieved. The speed of the particles ranges from about 5 to 20 m/s. An aerodynamic lens was used for single-particle diffraction of virus particles (Seibert et al., 2011), soot (Loh et al., 2012), particle dimers (Starodub et al., 2012), and cell organelles (Hantke et al., 2014). High particle hit rates have been achieved, although the fraction of those hits that occur in the most intense part of the focused beam is lower. Thus, the hit fraction depends on the X-ray beam profile in the focus, which tends to have wide low-intensity wings, and the threshold at which a pattern is counted as a hit. It might be feasible to inject sub-micron sized crystals by this approach, which could benefit in collecting data with less background at lower energies. Very recently, a compact aerosol injector was demonstrated that drives particles towards a focus of dimensions less than 10 lm diameter using a converging conical nozzle (Kirian et al., 2015). The injector is comparable in size to the gas-focused liquid jets described below, and hence could be interchangeably mounted with those. This method utilizes only the one orifice and thus gas is emitted at high speed, imparting velocities to the particles above 250 m/s. The higher the particle speed, the lower the particle hit rate for a given X-ray pulse rate (since the particle density is reduced), but this injector is currently the only one with high enough speeds for the 4.5 MHz burst rate of the European XFEL. D. Gas-focused liquid jets
The gas-focused dynamic virtual nozzle (GDVN) creates a jet of liquid that is typically a few microns in diameter moving at a velocity of several meters per second, injected into vacuum. Most SFX experiments to date have utilized GDVNs to deliver liquid suspensions of crystals to the beam. The nozzle consists of a hollow capillary usually made of glass and centered within a larger capillary tube. High-pressure gas flows through the interstitial space between the capillaries and focuses the liquid jet coming out of the inner capillary to a diameter that is much smaller than the orifice of that capillary (DePonte et al., 2008). This avoids clogging of
041709-8
Chavas, Gumprecht, and Chapman
Struct. Dyn. 2, 041709 (2015)
the delivered particles that would occur by simply using a small orifice without gas focusing. Jet diameters of 1–5 lm are typically achieved, depending on the capillary diameters, the nozzle design, and the liquid and gas flows. Jet diameters as small as 300 nm have been demonstrated with a modified design where the orifice of the liquid line occurs at the point of the gas expansion into vacuum (DePonte et al., 2009). The emitted liquid forms a cylindrical column that eventually breaks up into drops. The breakup can be driven at a fixed frequency so that droplets are timed to the X-ray pulse arrival (Shapiro et al., 2008). However, since the drop diameter is about twice the column diameter, a lower background is achieved by placing the X-ray focus in the column. Particles and crystals that are elongated tend to align their long axis in the direction of the flow in the column and lose that alignment in the breakup region. It has been the practice so far to run the jet continuously rather than pulsing it to match the X-ray repetition rate. As such, the liquid flow rate is typically greater than 10 ll min 1 and tens of milligrams of microcrystals are often required for a full data set. Photoactivated samples can be studied by laser-pumping the sample at the required time before the X-ray pulse arrival, which is usually done by illuminating the entire jet with the pump beam. However, for delays longer than about 100 ls, it may be necessary to illuminate the sample inside the nozzle (Kupitz et al., 2014). By introducing another liquid capillary inserted in the first liquid line, it is possible to make a mixing jet (Wang et al., 2014). Here, the sample can mix diffusively with a substrate in the laminar flow of the jet at a well-defined time before X-ray exposure, which can be varied by a clever telescopic design. GDVNs have also been made by applying the soft lithography techniques of microfluidics (Trebbin et al., 2014). This has the advantage of easier mass production and in employing many designs for mixing and pulsing of jets. E. Electrospun jets
Instead of gas focusing, electrospinning forms a small jet in vacuum or atmosphere by application of high voltage (between the liquid and an external electrode) that creates a Taylor cone. Samples can consist of particles embedded in a viscoelastic polymer or in a conductive liquid of the right viscosity (Sierra et al., 2012 and Bogan et al., 2008). Slower velocities than liquid jets can be attained, and voltage gives an extra degree of freedom that allows higher velocity for a given sample flow rate (producing sub-micron jets). Typical flow rates are 0.14 to 3.1 ll min 1. No effect of the high electric field on the sample has been observed in protein structures obtained using this delivery mechanism (Kern et al., 2013). It is worth to note that this method is not suitable for measurements with synchrotron radiation since it was observed (in unpublished experiments at the Swiss Light Source by Bogan and Chapman) that the X-ray beam disrupts the formation of the Taylor cone, possibly due to ionization of the rest gas. F. Extruded viscous flows
The LCP extrusion nozzle is similar to the GDVN except that the sample is embedded in a high-viscosity medium that is pushed out of a nozzle at high pressure (Weierstall et al., 2014). This has primarily been used to dispense membrane proteins, such as G-protein coupled receptors, that crystallize in LCP (Liu et al., 2013 and Weierstall, 2014). The diameter of the extruded material matches the nozzle diameter (about 20 to 50 lm). Coaxial gas is used to stabilize the flow direction, and the method works in vacuum or air (Botha et al., 2015 and Nogly et al., 2015). The velocity of the medium is much slower than that of liquid jets and can be varied between about 25 to 100 mm s 1. This conserves sample and with the 120 Hz repetition rate of LCLS it has been possible to run the jet at less than 100 lm between shots, essentially hitting every crystal that flows by with an X-ray FEL repetition rate of 60 Hz. At the European XFEL, it would only be possible to utilize such jets with a single X-ray pulse per pulse train, that is, at 10 Hz. Due to the required high pressure, the reservoir must be located close to the nozzle, but the reservoir volume only needs be
